The knowledge of complex three-dimensional folds of RNA structures is essential to understand the
increasing number of their biological functions. Both, X-ray crystallography and NMR spectroscopy,
are principal experimental methods to deliver RNA structural models at high resolution. However,
there is a great demand in the biological research community to envisage new, yet experimentally
inaccessible three-dimensional RNA structures to address particular questions on the structure-function
relationship. Here, we present RNAComposer (version 1.0), an advanced tool for modeling of RNA structures.
It's aim is to build atomic-resolution 3D models of large RNAs, based on their secondary structures,
using the RNA FRABASE dictionary relating
RNA secondary structure and tertiary structure elements.
The general idea of RNAComposer is based on the following processing path:

Interactive mode is mainly intended for those, who want to test the capabilities
of the system. It allows to run RNAComposer for a single sequence (up to 500 nt long)
and its secondary structure topology to build one output 3D model. A user is provided the information
about processing status in the real time. Pdb file with the generated model is available
for download immediately after the end of modeling. This file can be sent via email,
to the user request. In addition,
JSmol visualization of the model is provided on the webpage. More about this mode...

batch mode

In the batch mode, available for registered users, RNAComposer can compose of up to
1000 RNA structure 3D-models based on a set of 100 different secondary structures,
provided by single user at a time. Although, in principle, the method itself allows
to build models for RNA sequences of unlimited length, in the batch mode we have
set the server threshold to strands of up to 500 nt residues. The data entered by the
user as a single batch is queued in the system and served according to the queueing
algorithm. Pdb file(s) and log file(s) generated for the batch are available for
download immediately after the end of modeling and can be accessed from the user
workspace. According to the account settings
these files can be sent via email to the given address.
More about this mode...

Interactive mode is designed for users who want to predict a single structure for
a short sequence (up to 500 nts). Users are recommended to provide their own secondary
structure as input data. However, in this mode, it is possible to use one of the
applications for RNA secondary structure prediction which have been incorporated
into the RNAComposer pipeline.
A secondary structure introduced into RNAComposer define a single
task in the system. Processing a task in interactive mode results in generating
a single RNA 3D structure.

Input data in interactive mode define one task and consist of: task identifier,
sequence, and - optionally - secondary structure topology or application for secondary structure
prediction. Each information is provided in a separate line. Thus, a typical input should be
formatted in the following way:

Optionally, in any place of the entry, the user can insert a comment line starting from
"#".

First line indicates where task definition begins. It must be placed before the
specification of a sequence and a secondary structure topology. This line is followed by
the exact definition of the structure, in which user can specify:

only the sequence.

the sequence and the secondary structure topology.

the sequence and the application for RNA secondary structure prediction.

The secondary structure prediction method can be also selected from the list displayed after clicking Select secondary structure prediction method.
If the 3rd line of the entry remains empty and the method is not selected from the list, CentroidFold is applied by default.

a base pair is represented as a pair of opening (left) and closing (right) brackets,
i.e. "(" and ")".

In our system, this notation has been extended to represent secondary structure
of pseudoknots, by inserting squared "[" and "]" brackets for lower-order structures,
while the curly brackets "{" and "}" and angle brackets "<" and ">" are used for
higher and most complicated pseudoknots.

User should provide an input secondary structure topology encoded in dot-bracket. However,
we support also CT and BPSEQ notations by providing converters between
these formats and dot-bracket in the Tools page.

After running RNAComposer for a task, a user is taken to the task progress information
page, where the succeeding steps of structure modeling are announced. The information
about the result of each step and its computing time is released in the real time,
due to a progress of computation. Just after the end of modeling, a pdf file with
the resulting structure is available for download and visualized with JSmol.
The pdf can be sent via email to the address provided by the user with
entry data.

Three examples, ready to be loaded, are available to the user. Example 1 shows a
query which should be provided to model a structure of HIV-2 DIS RNA hairpin. Example
2 concerns encoding RNA structure with a pseudoknot, which is also supported by RNAComposer.
Example 3 presents the way to run RNAComposer when one does not have a secondary structure
and decides to use one of the tools
incorporated into the system.

The following tables show graphical and text (in the RNAComposer accepted format)
representation of each example input.

Batch mode is available only for registered users.
To enter this mode login to the system is required.
Batch mode has been designed for those who perform large-scale modeling. Input sequence
length in this mode is limited up to 500 nts. 1-10 secondary structures can be provided
for a single sequence (at least one is required). User is allowed to provide of up
to 10 sequences at a time. Such a multiple input is called a batch. One batch
is composed of tasks (a task is associated with one secondary structure). Processing a task
in the batch mode results in generating either one or multiple output (i.e. 1-10 RNA 3D
structures depending on the user requirements).

Batch can be either entered directly to the entry box or uploaded from a file. In
both cases the same data format must be followed. A batch should consist of 1-10
sections. Each section concerns one sequence and contains: an identifier, a sequence, and - optionally -
1-10 definitions of secondary structure topology associated with the same sequence. Each information should be provided
in a separate line. A single batch section should be formatted in the following way:

line

type

contents

1st line

obligatory

">" followed by a unique identifier

2nd line

obligatory

RNA sequence in the one-letter format

3rd-12th line

optional

secondary structure topologies in the dot-bracket notation or name of the application for secondary structure prediction
(one secondary structure or application name per line)

First line indicates where the section begins. It must be placed before the specification
of a sequence. The secondary structure topology - if provided - should be
encoded
in the dot-bracket notation. For those having their secondary structures in
CT or
BPSEQ,
appropriate converters to dot-bracket are available in the
Tools page. If neither secondary structure topology nor the name of application for secondary structure prediction is provided, the structure is predicted by CentroidFold.
Optionally, in any place of the batch, user can insert a comment line starting with
"#".

Characters accepted in the batch

The following characters are accepted in the batch entry box and batch file:

Three examples, ready to be loaded, are available to the user. Example 1 shows a
query which defines a structure of tetrahymena ribozyme (1X8W, chain B) with 247
nts log sequence. One secondary structure is provided in this example.
Example 2 presents the way to format a batch including several different secondary structure topologies
for one sequence. A 120 nts long sequence of 5S rRNA E.Coli taken from 2AWB (chain
A, mutant 12C>12A) serves as the example. Example 3 concerns runing RNAComposer
for a multi-sequence batch.

During preparation of 3D structure elements that compose the 3D model, respective elements are selected from the associated structural repository. User can define constraints (filters) to be applied during 3D elements search. Four filtering options are available after clicking the Filter 3D structure elements repository checkbox:

Exclude selected PDB structures
After clicking this checkbox, user can provide PDB identifiers (case-insensitive)
of all structures that should be dismissed during 3D structure elements search.
Every PDB id should be given in a separate line. Other accepted separators of identifiers
are ',' (comma) and ';' (semicolon). The list of identifiers
can be uploaded from file or entered directly to the entry box (the box is
displayed after clicking Exclude selected PDB structures).

Use X-ray determined structures only
If this option is selected, only these 3D structure elements are accepted by the search procedure
which derive from X-ray determined structures. Additionally, in the Set
resolution threshold entry box, user defines the maximum value of
resolution threshold (default = 3.0A) that should be satisfied by source X-ray structures (and thus, by
the 3D structure elements themselves).

Generate A-RNA-based single strands
If this option is selected, all single stranded fragments of the predicted 3D
structure are generated by NAB procedure (Case et al., 2015), based on the input
sequence and the template structure of A-RNA.

Generate A-RNA-based double helices
If this option is selected, all double helices within the predicted 3D
structure are generated by NAB procedure (Case et al., 2015), based on the input
sequence and the template structure of A-RNA.

User can restrain the process of 3D structure minimization by providing restraints for atom distances or torsion angles. This option is enabled if the user clicks Define 3D structure minimization restraints checkbox under batch entry box and selects one of checkboxes that appear thereafter:

In order to enter atom distance restraints one should fist click Define 3D structure minimization restraints, and next check Add atom distance restraints checkbox. This enables entering the data into a separate entry box.
Atom distance restraints can be either entered directly to the restraints entry
box or uploaded from a file. In both cases the same data format must be followed. User provides
a set of distance restraints concerning one or more secondary structures. More
than one set of restraints can be provided at a time. Subsequent sections containing
set descriptions should be separated by an empty line. A single section (set of restraints) should
be formatted as follows:

Sequence(s) and secondary structure topologie(s) for which atom distance restraints should be applied
are specified in the headline. Headline should have the following format:

<sequence numbers>;<secondary structure topology numbers>

where:

<sequence numbers>

is a list of serial numbers of sequences entered in
the batch entry box, separated by commas. At least one sequence number is required.
A maximum number of specified sequences is 10 (the same as in the batch entry box).
Sequences are indexed starting with 1.

<secondary structure topology numbers>

is a list of serial numbers of secondary structure topologies
defined for specified sequence(s), separated by commas. At least one structure number is required.
A maximum number of topologies is 10 (same as in the batch entry box, where at most
10 structures are accepted for one sequence).
Secondary structure topologies are indexed starting with 1 (separately for each sequence).

Examples of section headline:
1;2
distance restraints defined in the succeeding lines apply to the model(s) built by RNAComposer
for the 2nd secondary structure topology provided for the 1st sequence in the batch.

1;1,3
distance restraints defined in the succeeding lines apply to the models built by RNAComposer
for the 1st and 3rd secondary structure topologies provided for the 1st sequence in the batch.

1,2;1
distance restraints defined in the succeeding lines apply to the models built by RNAComposer
for the 1st secondary structure topology provided for the 1st sequence, and for the 1st secondary
structure topology provided for the 2nd sequence in the batch.

2,3;1,3,4
distance restraints defined in the succeeding lines apply to the models built by RNAComposer
for the 1st, 3rd and 4th secondary structure topology provided for the 1st sequence,
the 1st, 3rd and 4th secondary structure topology defined for the 3rd sequence,
and for the 1st, 3rd and 4th secondary structure topology specified under the 4th sequence in the batch.

distance restraints line

Atom distance restraints are specified under the headline, one line per restraint.
Distance restraints line should have the following format:

<residue 1> <atom 1>
<residue 2> <atom 2>
<distance> <minus> <plus>

i.e. it contains 7 fields, separated by single spaces and having
the following meaning:

field

meaning

format

<residue 1><residue 2>

serial number of the 1st residueserial number of the 2nd residue

max 4 charactersmax 4 characters

<atom 1><atom 2>

name of the atom from the 1st residuename of the atom from the 2nd residue

First set of restraints will be applied to the models built for the 1st and the 2nd secondary structure topologies defined for the 1st sequence in the batch.
Second set of restraints will be applied to the models built for the 1st secondary structure topologies defined for the 3rd and 4th sequence in the batch.

In order to enter torsion angle restraints one should fist click Define 3D structure minimization restraints, and next check Torsion angle restraints checkbox. This enables entering the data into a separate entry box.
Torsion angle restraints can be either entered directly to the restraints entry
box or uploaded from a file. In both cases the same data format must be followed. User provides
a set of angle restraints concerning one or more secondary structures. More
than one set of restraints can be provided at a time. Subsequent sections containing
set descriptions should be separated by an empty line. A single section (set of restraints) should be formatted as follows:

Sequence(s) and secondary structure topologie(s) for which torsion angle restraints should be applied are specified in the headline. Headline should have the following format:

<sequence numbers>;<secondary structure topology numbers>

where:

<sequence numbers>

is a list of serial numbers of sequences entered in
the batch entry box, separated by commas. At least one sequence number is required.
A maximum number of specified sequences is 10 (the same as in the batch entry box).
Sequences are indexed starting with 1.

<secondary structure topology numbers>

is a list of serial numbers of secondary structure topologies
defined for specified sequence(s), separated by commas. At least one structure number is required.
A maximum number of topologies is 10 (same as in the batch entry box, where at most
10 structures are accepted for one sequence).
Secondary structure topologies are indexed starting with 1 (separately for each sequence).

Examples of section headline:
1;2
torsion angle restraints defined in the succeeding lines apply to the model(s) built by RNAComposer
for the 2nd secondary structure topology provided for the 1st sequence in the batch.

1;1,3
torsion angle restraints defined in the succeeding lines apply to the models built by RNAComposer
for the 1st and 3rd secondary structure topologies provided for the 1st sequence in the batch.

1,2;1
torsion angle restraints defined in the succeeding lines apply to the models built by RNAComposer
for the 1st secondary structure topology provided for the 1st sequence, and for the 1st secondary
structure topology provided for the 2nd sequence in the batch.

2,3;1,3,4
torsion angle restraints defined in the succeeding lines apply to the models built by RNAComposer
for the 1st, 3rd and 4th secondary structure topology provided for the 1st sequence,
the 1st, 3rd and 4th secondary structure topology defined for the 3rd sequence,
and for the 1st, 3rd and 4th secondary structure topology specified under the 4th sequence in the batch.

torsion angle restraints line

Torsion angle restraints are specified under the headline, one line per restraint.
Torsion angle restraints line should have the following format:

In the batch mode user has the possibility to interfere in selection of the 3D structure elements that compose the final model. In particular, this option allows the user to replace 3D structure elements selected by the automated routine of RNAComposer. When RNAComposer finishes computation for user input data, it provides the final 3D model and a log file with the results of intermediate computational steps. The log file enumerates i.a. (i) secondary structure elements
resulting from input data fragmentation (identifier, sequence and dot-bracket-encoded secondary
structure topology are given for every element), and (ii) 3D structure elements associated with
their secondary structure counterparts. The user can decide to modify selected parts of the obtained 3D model by providing own 3D structure elements and indicating which elements of originally predicted 3D structure should be substituted.
To do this, one should click Insert
own 3D structure elements button and, next, introduce own 3D structure element(s) in PDB
format, preceded by the identifier(s) of associated secondary structure element(s). More than one
structure element can be substituted at a time. A definition of substitution section should be formatted as follows:

log file with the information about task processing phases and results of each phase,

pdf file with a generated structure.

After running RNAComposer for a batch, a user can see the progress within the User workspace page.
When the batch is finished the results are available for download in a zip file.
The file can be sent via email to the address provided by the user in the Account settings.

Registration is required for those users who want to run RNAComposer in the batch mode.
Interactive mode is available without registration.
To register one should create an account (click Create an account link on the gray area under the main menu). Registration requires
providing an email, username (user ID) and password. Note that username must be unique within the system. Check availability button is
placed on the registration webpage to help users define a unique ID.
After the account data are submitted to the system (Create an account button clicked), a verification email is sent to the user.
Registration is completed when the user clicks the link given in the email. The account is available immediately after verification.

Once logged into RNAComposer system, user can change account settings including email address and password (to do so select
Account settings link from user menu in gray area). The decision on ones datasets (i.e. files including the results of
batch processing by RNAComposer) can be also set here. By default, user is notified via email when the results are available
(Send email notification without attachements is checked in the Account settings page).
The result files are stored in the system for 14 days, available for download from User workspace.
The files may be attached to the notification email on user request (check Send email notification with result files attached
to activate this option).

Logged in user can access his workspace by selecting My workspace from user menu in gray area. The following
information is available from the workspace:

a number of user's batches uploaded to the system,

detailed information about each batch including: batch identifier, number of sequences in the batch,
day & time of an upload, launch and completion by RNAComposer engine,

result files for completed batches ready for download.

To access the information about secondary structures defined in a single batch its identifier
should be clicked.

User can delete uploaded batches by selecting a batch (checkbox on the leftmost column should be checked for a batch) and
clicking Delete selected button. If not deleted by the user, batches are removed from the system 14 days after completion.

Tools page provides support to the users having their RNA secondary
structure encoded in CT (Connect) or BPSEQ format. Since RNAComposer
accepts only dot-bracket notation, we offer an interface to
CT→dot-bracket and BPSEQ→dot-bracket converters. They are
user-friendly shortcuts to functions available in RNApdbee.

Secondary structure in BPSEQ or CT format can be uploaded directly
from a file. After conversion, user can save dot-bracket secondary
structure into a file ('Save') or send it to the task entry box
('Send to homepage'), where it can be immediately used to run new
task.

RNAComposer maintains an official mirror site available at http://rnacomposer.cs.put.poznan.pl. Please, use RNAComposer mirror if the current site is overloaded, your job processing time is irrationally long, if you observe unstable system behaviour or network disruption problems.